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In this work, we investigate the optical properties variation of thermally grown W oxides during the exposure to low energy deuterium (D) plasma. In-situ ellipsometry in the 400–1000 nm range was coupled to ex-situ diagnostics, such as X-ray photoelectron spectroscopy (XPS) and focused ion beam coupled to scanning electron microscopy (FIB-SEM), to probe the evolution of chemical and morphological properties of the W oxides. First, a 70 nm thick WO3 layer was exposed to D plasma at a surface temperature of 650 K. An important reduction of the oxide layer was observed by FIB-SEM, and in-situ ellipsometry showed the evolution of the optical constants n and k of the oxide towards the ones of pure W metal. Secondly, a 300 nm thick WO3 layer was exposed to D plasma at a surface temperature below 373 K. In order to follow the oxide evolution step by step, we alternated in-situ ellipsometry and XPS surface characterization. A quite similar evolution of the optical constants was observed, in particular an increase of the extinction coefficient k in the near infrared, which was linked to a progressive reduction of the oxidation level at the surface, as seen by XPS. Interestingly, the reduction of the oxide at 373 K was below the resolution of the FIB-SEM. This observation indicates that ellipsometry in the 400–1000 nm range is able to follow in-situ the reduction of WO3 oxides by D plasma with high surface sensitivity.

One of the engineering challenges in developing more reliable electrodes in solid oxide fuel cells is to reduce residual stresses generated during electrode fabrication. The Young's modulus of porous ceramic materials employed in the electrodes is a fundamental property essential for evaluating the residual stresses of electrodes. In this study, the correlation between Young's modulus and the microstructure of the cathode material La0.6Sr0.4Co0.2Fe0.8O3-δ was evaluated numerically using a voxel-based finite element method in conjunction with the three-dimensional mesoscale techniques, including a kinetic Monte Carlo and a discrete element method. The results of simulations utilizing the FIB-SEM reconstructed microstructures are in good agreement with those of micro-indentation experiments, indicating that our methodology is effective for estimating the Young's modulus of porous ceramics at room temperature. Furthermore, we reveal that the porosity dependence of Young’s modulus can be well described by an empirical power law based on the percolation model. In particular, our findings indicate that the dependencies on porosity are highly related to the homogeneity of the initial powder microstructures before sintering. Finally, our results suggest that the effective Young’s modulus of the porous materials at any porosity has a correlation with the tortuosity factor.

Abstract Magnetic signals in igneous rocks arise from assemblages of iron‐oxide bearing minerals that differ in for example, size, shape, and chemistry. Paleomagnetic measurements on bulk samples measure millions of such grains simultaneously, producing a statistical ensemble of the magnetic moments of the individual grains. Scanning magnetometry techniques such as the Quantum Diamond Microscope (QDM) measure magnetic signals on micrometer scales, allowing the identification of magnetic moments of individual grains in a sample using for example, Micromagnetic Tomography (MMT). Here we produce a grain‐size distribution of iron‐oxides in a typical Hawaiian basalt from the superparamagnetic threshold (∼40 nm) to grains with a diameter of 10 µm. This grain‐size distribution is obtained by combining FIB‐SEM and MicroCT data from sister specimens, and normalizing them to the mineral surface area of non‐magnetic minerals. Then we use this grain‐size distribution to determine the contributions of individual magnetic carriers to bulk magnetic measurements and surface magnetometry. We found that measurements on bulk samples are sensitive to relatively small grain sizes in the realm of single domain or vortex states (1 µm. This implies that bulk measurements cannot be compared straightforwardly to signals from surface magnetometry from the same sample. Moreover, our observations explain why MMT results are insensitive to the presence of many small grains in a sample that intuitively should hamper their outcome.

The brain contains thousands of millions of synapses, exhibiting diverse structural, molecular, and functional characteristics. However, synapses can be classified into two primary morphological types: Gray’s type I and type II, corresponding to Colonnier’s asymmetric (AS) and symmetric (SS) synapses, respectively. AS and SS have a thick and thin postsynaptic density, respectively. In the cerebral cortex, since most AS are excitatory (glutamatergic), and SS are inhibitory (GABAergic), determining the distribution, size, density, and proportion of the two major cortical types of synapses is critical, not only to better understand synaptic organization in terms of connectivity, but also from a functional perspective. However, several technical challenges complicate the study of synapses. Potassium ferrocyanide has been utilized in recent volume electron microscope studies to enhance electron density in cellular membranes. However, identifying synaptic junctions, especially SS, becomes more challenging as the postsynaptic densities become thinner with increasing concentrations of potassium ferrocyanide. Here we describe a protocol employing Focused Ion Beam Milling and Scanning Electron Microscopy for studying brain tissue. The focus is on the unequivocal identification of AS and SS types. To validate SS observed using this protocol as GABAergic, experiments with immunocytochemistry for the vesicular GABA transporter were conducted on fixed mouse brain tissue sections. This material was processed with different concentrations of potassium ferrocyanide, aiming to determine its optimal concentration. We demonstrate that using a low concentration of potassium ferrocyanide (0.1%) improves membrane visualization while allowing unequivocal identification of synapses as AS or SS.

ATG9A is an important membrane protein in mammalian macroautophagy. The formation of autophagosomes and phagophores is blocked in atg9a KO cells. However, it remains possible that residual membrane formation activity exists in these cells. These precursor structures that precede phagophores are, if they exist, rare and may be difficult to find. Here, we introduce the modified volume correlative light and electron microscopy (CLEM) method to analyze these structures three-dimensionally. In addition to target proteins, mitochondria were labeled as a landmark for precise correlation of slice images by a confocal fluorescence microscope and a focused ion beam scanning electron microscope. We found phagophores and small membrane vesicles near SQSTM1/p62 aggregates in atg9a KO cells, indicating that phagophores could be formed in atg9a-deficient cells, although they were immature and inefficient. Furthermore, we found that RB1CC1/FIP200-positive structures formed clusters around SQSTM1/p62 with ferritin and TAX1BP1. Taken together, our method contributes to the understanding of undiscovered fine structures. Abbreviations: CLEM: correlative light and electron microscopy; EM: electron microscopy; ER: endoplasmic reticulum; FIB-SEM: focused ion beam scanning electron microscopy; FM: fluorescence microscopy; GFP: green fluorescent protein; KO: knock out; MEF: mouse embryonic fibroblast; PBS: phosphate-buffered saline; ROI: region of interest; SEM: scanning electron microscopy.

Unicellular euglyphid testate amoeba Paulinella micropora with filose pseudopodia secrete approximately 50 siliceous scales into the extracellular template-free space to construct a shell isomorphic to that of its mother cell. This shell-constructing behavior is analogous to building a house with bricks, and a complex mechanism is expected to be involved for a single-celled amoeba to achieve such a phenomenon; however, the three-dimensional (3D) structure of the shell and its assembly in P. micropora are still unknown. In this study, we aimed to clarify the positional relationship between the cytoplasmic and extracellular scales and the structure of the egg-shaped shell in P. micropora during shell construction using focused ion beam scanning electron microscopy (FIB-SEM). 3D reconstruction revealed an extensive invasion of the electron-dense cytoplasm between the long sides of the positioned and stacked scales, which was predicted to be mediated by actin filament extension. To investigate the architecture of the shell of P. micropora, each scale was individually segmented, and the position of its centroid was plotted. The scales were arranged in a left-handed, single-circular ellipse in a twisted arrangement. In addition, we 3D printed individual scales and assembled them, revealing new features of the shell assembly mechanism of P. micropora. Our results indicate that the shell of P. micropora forms an egg shape by the regular stacking of precisely designed scales, and that the cytoskeleton is involved in the construction process.

Flexible graphite (FG) with ρ = 1 g/cm3 density is a type of highly porous and anisotropic graphite, mainly used for gaskets and sealing applications, but also suitable for energy absorption, such as in the beam dumping devices of the Large Hadron Collider (see Heredia 2021 [1]). Knowledge of its microstructure and mechanical properties needs to be developed for the selection of an adequate material model able accurately predict stresses and failure in FG components. Here, the FG microstructure properties available in literature are reviewed, followed by Focused Ion Beam - Scanning Electron Microscopy investigation and compression tests. Specifically, a single 100 μm × 150 μm cross section was obtained, and the 2D pore sizes and shapes were quantified using image segmentation. Monotonic and cyclic out-of-plane compression tests were performed in single and stacked configuration. Stress-strain curves showed three domains: the initial toe, the transition and the densification domain. The cyclic tangent modulus was also calculated from the cyclic tests. Many observations suggested that FG behaves similarly to crushable foams, crumpled materials and compacted powders, and that both crystalline microstructure and crumpled mesostructure play a predominant role in the deformation mechanism.

FlowDenoising is a software tool that implements an adaptive Gaussian denoising filter that preserves visually appreciable structures in volumes of 3D electron microscopy (3DEM). It proceeds by nonrigidly aligning the 2D slices in each dimension, using an optical flow estimator, prior to applying a standard separable (1D) Gaussian filter. FlowDenoising has been developed in Python leveraging well-known public domain libraries, such as OpenCV and NumPy. Furthermore, the software tool exploits data-level parallelism to significantly reduce processing times. Its abilities to denoise huge volumes in just minutes on standard multicore computers makes it a useful tool in 3DEM to explore the interior of cells and tissues at the nanoscale.

Abstract Porous carbon‐based electrodes are frequently applied in electrochemical energy technologies, for instance in fuel cells and redox flow batteries. In previous work, we observed that the final structure of a fuel cell electrode is dominated by both the morphology of the support material and its processing into a 3D porous structure. Herein, the impact of catalyst support morphology on the performance of polymer electrolyte membrane fuel cells was studied comparing carbon‐supported platinum catalysts only differing in the shape of the carbon support material with otherwise similar features. Carbon‐supported Pt catalysts were obtained by carbonization of polyaniline (PANI) in long fibrous, short fibrous, and granular shape. The chemical identity of the PANI precursors was demonstrated by FTIR spectroscopy and elemental analysis (EA). The final carbon‐supported platinum catalysts were characterized by EA, Raman spectroscopy, XRD, and TEM exhibiting similar degree of carbonization, nanoparticle size, and nanoparticle dispersion. The effect of support morphology and the resulting differences in the 3D structure of the porous electrode were investigated by focused ion beam‐scanning electron microscopy slice and view technique and correlated to their fuel cell performance.

Abnormal tendons are rarely ever repaired to the natural structure and morphology of normal tendons. To better guide the repair and regeneration of injured tendons through a tissue engineering method, it is necessary to have insights into the internal morphology, organization, and composition of natural tendons. This review summarized recent researches on the structure and function of the extracellular matrix (ECM) components of tendons and highlight the application of multiple detection methodologies concerning the structure of ECMs. In addition, we look forward to the future of multi-dimensional biomaterial design methods and the potential of structural repair for tendon ECM components. In addition, focus is placed on the macro to micro detection methods for tendons, and current techniques for evaluating the extracellular matrix of tendons at the micro level are introduced in detail. Finally, emphasis is given to future extracellular matrix detection methods, as well as to how future efforts could concentrate on fabricating the biomimetic tendons.

Deriving the detailed synaptic connections of an entire nervous system is the unrealized goal of the nascent field of connectomics. For the fruit fly Drosophila, in particular, we need to dissect the brain, connectives, and ventral nerve cord as a single continuous unit, fix and stain it, and undertake automated segmentation of neuron membranes. To achieve this, we designed a protocol using progressive lowering of temperature dehydration (PLT), a technique routinely used to preserve cellular structure and antigenicity. We combined PLT with low temperature en bloc staining (LTS) and recover fixed neurons as round profiles with darkly stained synapses, suitable for machine segmentation and automatic synapse detection. Here we report three different PLT-LTS methods designed to meet the requirements for FIB-SEM imaging of the Drosophila brain. These requirements include: good preservation of ultrastructural detail, high level of en bloc staining, artifact-free microdissection, and smooth hot-knife cutting to reduce the brain to dimensions suited to FIB-SEM. In addition to PLT-LTS, we designed a jig to microdissect and pre-fix the fly’s delicate brain and central nervous system. Collectively these methods optimize morphological preservation, allow us to image the brain usually at 8 nm per voxel, and simultaneously speed the formerly slow rate of FIB-SEM imaging.

This study reports the characterisation of human dental enamel caries using synchrotron nanoscale correlative ptychography and spectroscopic mapping in combination with scanning electron microscopy. A lamella ̴2.4 µm thick was extracted from a carious enamel region of a tooth using focused ion beam-scanning electron microscopy and transferred to two synchrotron beamlines to perform hard X-ray nano-fluorescence spectroscopy simultaneously with differential phase contrast mapping at a beam size of 55 × 45 nm. Soft X-ray ptychography data was then reconstructed with a pixel size of 8 nm. The two dimensional variation in chemistry and structure of carious enamel was revealed at the nanoscale, namely, the organisation of hydroxyapatite nano-crystals within enamel rods was imaged together with the inter-rod region. Correlative use of electron and X-ray scanning microscopies for the same sample allowed visualisation of the connection between structure and composition as presented in a compound image where colour indicates the relative calcium concentration in the sample, as indicated by the calcium Kα fluorescence intensity and grey scale shows the nanostructure. This highlights the importance of advanced correlative imaging to investigate the complex structure-composition relationships in nanomaterials of natural or artificial origin.

Cryogenic electron tomography (cryo-ET) combined with subtomogram averaging, allows in situ visualization and structure determination of macromolecular complexes at subnanometre resolution. Cryogenic focused ion beam (cryo-FIB) micromachining is used to prepare a thin lamella-shaped sample out of a frozen-hydrated cell for cryo-ET imaging, but standard cryo-FIB fabrication is blind to the precise location of the structure or proteins of interest. Fluorescence-guided focused ion beam (FIB) milling at target locations requires multiple sample transfers prone to contamination, and relocation and registration accuracy is often insufficient for 3D targeting. Here, we present in situ fluorescence microscopy-guided FIB fabrication of a frozen-hydrated lamella to address this problem: we built a coincident three-beam cryogenic correlative microscope by retrofitting a compact cryogenic microcooler, custom positioning stage, and an inverted widefield fluorescence microscope (FM) on an existing FIB scanning electron microscope. We show FM controlled targeting at every milling step in the lamella fabrication process, validated with transmission electron microscope tomogram reconstructions of the target regions. The ability to check the lamella during and after the milling process results in a higher success rate in the fabrication process and will increase the throughput of fabrication for lamellae suitable for high-resolution imaging.

Abstract This paper discusses the relationship between the elongation and compression behavior and microstructural changes under redox cycles of porous Ni(O)–YSZ cermets for solid oxide fuel cells (SOFC). Mechanical damage in SOFC and SOEC is one of the most important degradation factors governing the electrical performance of cells. Therefore, it is necessary to know the mechanical properties of each component material, such as elastic and deformation properties, in the operating environment. Particularly, of the Ni(O)–YSZ cermets which currently makes up 90% of the volume of the cell, with present mainstream anode supported SOFC and SOEC. Therefore, understanding the properties of the Ni(O)–YSZ cermets plays an important role in ensuring the performance of the entire SOFC and SOEC. In this study, the microstructural changes of Ni(O)–YSZ cermet by reduction, re-oxidation and re-reduction were observed in detail using microstructural observations and systematically compared with the dimensional change behavior. For the dimensional change behavior, a simple model considering the initial porosity and Ni content is proposed, which successfully predicts the dimensional change due to re-oxidation. Furthermore, Ni(O)–YSZ cermets with high Ni content show large initial dimensional changes, but the dimensional reversibility improves with increase of the number of redox cycles.

The pore structures of shale in the Shahejie formation of Dongying depression were quantitatively characterized by FIB-SEM three-dimensional imaging technology. FIB-SEM was used to obtain high-resolution SEM images and through image registration, geometric correction and image segmentation, the organic pores and inorganic pores in the shale were distinguished. The pore space was converted into a structured pore network model via maximal ball method. Then, the pore size distributions and volume contributions of shale organic pores and inorganic pores, and the coordination number of the total pore network model were statistically analyzed via the pore network model. The results showed that due to the low thermal maturity of organic matter in the Shahejie formation, fewer organic pores were observed in the shale samples, and the number of pores was dominated by inorganic pores. Statistical analysis of the pore network model indicated that the studied shale samples considerably included mesopore (size of 2–50 nm) which accounted for 61.1%, but their contribution to the total pore volume was small (4.2%). Macropores (>50 nm) accounted for a relatively small proportion (38.9%) in number, but they provided the main storage space (95.8%) for the shale oil and gas. The volume contribution of pores to the total pore space at a certain bin size is controlled by the combination of pore number and the pore size. The coordination numbers of the two samples were mainly 0 and 1, indicating that the pore connectivity was poor and isolated pores accounted for most part. These results are critical for further reliable petrophysical simulations based on shale digital rocks as well as for the accurate understanding of their petrophysical properties.

Natural graphite ores are usually upgraded by froth flotation. However, complex processes with multistage grinding and flotation are required to achieve decent liberation and separation of graphite and gangue minerals. This study reports a short and improved flotation process for fine flake graphite ore by employing a thickening stage. The results indicated that increasing the regrinding concentration via thickening can improve the grinding efficiency and, thus, shorten the separation process. With thickening, a high-grade intermediate concentrate of 96.01% was obtained after three steps of cleaner flotation, which is close to the final concentrate after five steps. Particle size distribution analysis and FIB-SEM-EDS studies suggested that the main contribution of thickening–regrinding was to achieve better abrasion rather than attrition of the graphite flakes, thus liberating the interlayer impurities without reducing the size of the graphite flakes. This study offers a more cost-effective pathway for the simplified flotation of natural graphite ores.

Freshwater lakes are often polluted with various heavy metals in the Anthropocene. The iron-oxidizing microorganisms and their mineralized products can coprecipitate with many heavy metals, including Al, Zn, Cu, Cd, and Cr. As such, microbial iron oxidation can exert a profound impact on environmental remediation. The environmental pH is a key determinant regulating microbial growth and mineralization and then influences the structure of the final mineralized products of anaerobic iron-oxidizing bacteria. Freshwater lakes, in general, are neutral-pH environments. Understanding the effects of varying pH on the mineralization of iron-oxidizing bacteria under neutrophilic conditions could aid in finding out the optimal pH values that promote the coprecipitation of heavy metals. Here, two typical neutrophilic Fe(II)-oxidizing bacteria, the nitrate-reducing Acidovorax sp. strain BoFeN1 and the anoxygenic phototrophic Rhodobacter ferrooxidans strain SW2, were selected for studying how their growth and mineralization response to slight changes in circumneutral pH. By employing focused ion beam/scanning electron microscopy (FIB–SEM) and transmission electron microscopy (TEM), we examined the interplay between pH changes and anaerobic iron-oxidizing bacteria and observed that pH can significantly impact the microbial mineralization process and vice versa. Further, pH-dependent changes in the structure of mineralized products of bacterial iron oxidation were observed. Our study could provide mechanical insights into how to manipulate microbial iron oxidation for facilitating remediation of heavy metals in the environment.

The structural complexity of nervous tissue makes it very difficult to unravel the connectivity between neural elements at different scales. Numerous methods are available to trace long-range projections at the light microscopic level, and to identify the actual synaptic connections at the electron microscopic level. However, correlating mesoscopic and nanoscopic scales in the same cell, cell population or brain region is a problematic, laborious and technically demanding task. Here we present an effective method for the 3D reconstruction of labeled subcellular structures at the ultrastructural level, after single-neuron labeling in fixed tissue. The brain is fixed by intracardial perfusion of aldehydes and thick vibratome sections (250 μm) are obtained. Single cells in these vibratome sections are intracellularly injected with horseradish peroxidase (HRP), so that the cell body and its processes can be identified. The thick sections are later flat-embedded in epoxy resin and re-sectioned into a series of thinner (7 μm) sections. The sections containing the regions of interest of the labeled cells are then imaged with automated focused ion beam milling and scanning electron microscopy (FIB-SEM), acquiring long series of high-resolution images that can be reconstructed, visualized, and analyzed in 3D. With this methodology, we can accurately select any cellular segment at the light microscopic level (e.g., proximal, intermediate or distal dendrites, collateral branches, axonal segments, etc.) and analyze its synaptic connections at the electron microscopic level, along with other ultrastructural features. Thus, this method not only facilitates the mapping of the synaptic connectivity of single-labeled neurons, but also the analysis of the surrounding neuropil. Since the labeled processes can be located at different layers or subregions, this method can also be used to obtain data on the differences in local synaptic organization that may exist at different portions of the labeled neurons.

The egg plays a pivotal role in the reproduction of our species. Nevertheless, its fundamental biology remains elusive. Transmission electron microscopy is traditionally used to inspect the ultrastructure of female gametes. However, two-dimensional micrographs contain only fragmentary information about the spatial organization of the complex oocyte cytoplasm. Here, we employed the Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) to explore human oocyte intracellular morphology in three dimensions (3D). Volume reconstruction of generated image stacks provided an unprecedented view of ooplasmic architecture. Organelle distribution patterns observed in nine donor oocytes, representing three maturational stages, documented structural changes underlying the process by which the egg acquires developmental competence. 3D image segmentation was performed to extract information about distinct organelle populations, and the following quantitative analysis revealed that the mitochondrion occupies ∼ 4.26% of the maturing oocyte cytoplasm. In summary, this proof-of-concept study demonstrates the potential of large volume electron microscopy to study rare samples of delicate female gametes and paves the way for applying the FIB-SEM technique in human oocyte research.

At the end of the first larval stage, the nematode Caenorhabditis elegans developing in harsh environmental conditions is able to choose an alternative developmental path called the dauer diapause. Dauer larvae exhibit different physiology and behaviors from non-dauer larvae. Using focused ion beam-scanning electron microscopy (FIB-SEM), we volumetrically reconstructed the anterior sensory apparatus of C. elegans dauer larvae with unprecedented precision. We provide a detailed description of some neurons, focusing on structural details that were unknown or unresolved by previously published studies. They include the following: (1) dauer-specific branches of the IL2 sensory neurons project into the periphery of anterior sensilla and motor or putative sensory neurons at the sub-lateral cords; (2) ciliated endings of URX sensory neurons are supported by both ILso and AMso socket cells near the amphid openings; (3) variability in amphid sensory dendrites among dauers; and (4) somatic RIP interneurons maintain their projection into the pharyngeal nervous system. Our results support the notion that dauer larvae structurally expand their sensory system to facilitate searching for more favorable environments.

In the present study, solid oxide cells (SOCs) were operated in the electricity generation (solid oxide fuel cell, SOFC) mode and the hydrogen production (solid oxide electrolysis cell, SOEC) mode. The fuel electrodes fabricated with nickel-gadolinia doped ceria (Ni-GDC) and nickel-yttria stabilized zirconia (Ni-YSZ) composites were investigated. The correlations between changes in the microstructure and degradation of electrochemical performance are discussed. The degradation mechanisms correlated with Ni phase were found to be similar for Ni-GDC and Ni-YSZ electrodes. On the other hand, the stability of the ceramic phase differs significantly between the two electrode materials.

ABSTRACT Sabrina Absalon works in the field of cellular and molecular biology of Plasmodium falciparum, the most virulent parasite causing malaria in humans. In this mSphere of Influence article, she reflects on how the paper “3D nuclear architecture reveals coupled cell cycle dynamics of chromatin and nuclear pores in the malaria parasite Plasmodium falciparum” by Allon Weiner et al. (A. Weiner, N. Dahan-Pasternak, E. Shimoni, V. Shinder, et al., Cell Microbiol 13:967–977, 2011, https://doi.org/10.1111/j.1462-5822.2011.01592.x) triggered her aspiration to study the molecular mechanisms governing nuclear envelope assembly and integrity of P. falciparum throughout the intraerythrocytic development cycle.

The advantage of using porous materials for biofuel cells and biosensors is their very large internal surface area (where electron exchange takes place) compared to the overall material volume, yielding much larger current densities than on a bare solid electrode of the same size. However, limitations occur because of mass transfer resistance through the pores. We describe here a bottom-up approach to optimize the design of such materials, through the analysis and modeling of their porous structure. Electrodes prepared by replicating stacked Langmuir-Blodgett films, with 1-μm diameter interconnected spherical pores, were studied. Since pore window dimensions are around 100 nm, Focused Ion Beam-Scanning Electron Microscopy (FIB-SEM) has been performed to obtain a 3D reconstruction of the porous medium. Then, a determination of the geometrical characteristics has been achieved through image analysis. The structure of the sphere packing, the shape and size of the connections between spheres, the distances between spheres, the sphere diameters and the specific surface area have been analyzed. The porous medium is close to a face-centered cubic arrangement of spherical pores, but several deviations from ideality are present: missing pores (point defects), stacking errors (dislocations), and incomplete connection between spheres (only 50% of the ideal sphere connections are present). The consequence of such defects on transport is studied through image-based simulations of mass diffusion in the actual porous medium and in similar ideal media.

ABSTRACT The determination of the exact location of a protein in the cell is essential to the understanding of biological processes. Here, we report for the first time the visualization of a protein of interest in Saccharomyces cerevisiae using focused ion beam scanning electron microscopy (FIB-SEM). As a proof of concept, the integral endoplasmic reticulum (ER) membrane protein Erg11 has been C-terminally tagged with APEX2, which is an engineered peroxidase that catalyzes an electron-dense deposition of 3,3′-diaminobenzidine (DAB), as such marking the location of the fused protein of interest in electron microscopic images. As DAB is unable to cross the yeast cell wall to react with APEX2, cell walls have been partly removed by the formation of spheroplasts. This has resulted in a clear electron-dense ER signal for the Erg11 protein using FIB-SEM. With this study, we have validated the use of the APEX2 tag for visualization of yeast proteins in electron microscopy. Furthermore, we have introduced a methodology that enables precise and three-dimensional (3D) localization studies in yeast, with nanometer resolution and without the need for antibody staining. Because of these properties, the described technique can offer valuable information on the molecular functions of studied proteins. IMPORTANCE With this study, we have validated the use of the APEX2 tag to define the localization of proteins in the model yeast S. cerevisiae. As such, FIB-SEM can identify the exact 3D location of a protein of interest in the cell with nanometer-scale resolution. Such detailed imaging could provide essential information on the elucidation of various biological processes. APEX2, which adds electron density to a fused protein of interest upon addition of the substrate DAB, originally was used in mammalian studies. With this study, we expand its use to protein localization studies in one of the most important models in molecular biology.

High-resolution 3D images of organelles are of paramount importance in cellular biology. Although light microscopy and transmission electron microscopy (TEM) have provided the standard for imaging cellular structures, they cannot provide 3D images. However, recent technological advances such as serial block-face scanning electron microscopy (SBF-SEM) and focused ion beam scanning electron microscopy (FIB-SEM) provide the tools to create 3D images for the ultrastructural analysis of organelles. Here, we describe a standardized protocol using the visualization software, Amira, to quantify organelle morphologies in 3D, thereby providing accurate and reproducible measurements of these cellular substructures. We demonstrate applications of SBF-SEM and Amira to quantify mitochondria and endoplasmic reticulum (ER) structures.

Abstract Background Molecular and structural information on avian Trypanosoma spp. throughout Australia is limited despite their intrinsic value in understanding trypanosomatid evolution, diversity, and structural biology. In Western Australia tissue samples (n = 429) extracted from 93 birds in 25 bird species were screened using generic PCR primers to investigate the diversity of Trypanosoma spp. To investigate avian trypanosome structural biology the first 3-dimensional ultrastructural models of a Trypanosoma spp. (Trypanosoma sp. AAT) isolated from a bird (currawong, Strepera spp.) were generated using focussed ion beam milling combined with scanning electron microscopy (FIB-SEM). Results Here, we confirm four intercontinental species of avian trypanosomes in native Australian birds, and identify a new avian Trypanosoma. Trypanosome infection was identified in 18 birds from 13 different bird species (19%). A single new genotype was isolated and found to be closely related to T. culicavium (Trypanosoma sp. CC2016 B002). Other Trypanosoma spp. identified include T. avium, T. culicavium, T. thomasbancrofti, Trypanosoma sp. TL.AQ.22, Trypanosoma sp. AAT, and an uncharacterised Trypanosoma sp. (group C-III sensu Zidková et al. (Infect Genet Evol 12:102-112, 2012)), all previously identified in Australia or other continents. Serially-sectioning Trypanosoma sp. AAT epimastigotes using FIB-SEM revealed the disc-shaped kinetoplast pocket attached perpendicular to the branching mitochondrion. Additionally, the universal minicircle sequence within the kinetoplast DNA and the associated binding protein were determined in Trypanosoma sp. AAT. Conclusions These results indicate that bird trypanosomes are relatively conserved across continents, while being locally diverse, which supports the hypothesis that bird trypanosomes exist as fewer species than described in the literature. Evidence exists that avian Trypanosoma spp. are infecting mammals and could be transmitted by haemadipsid leeches. Trypanosoma sp. AAT is most likely a separate species currently found only in Australia and the first 3-dimentional ultrastructural analysis of an avian trypanosome provides interesting information on their morphology and organelle arrangement.

Serial-section electron microscopy such as FIB-SEM (focused ion beam scanning electron microscopy) has become an important tool for neuroscientists to trace the trajectories and global architecture of neural circuits in the brain, as well as to visualize the 3D ultrastructure of cellular organelles in neurons. In this study, we examined 3D features of mitochondria in electron microscope images generated from serial sections of four regions of mouse brains: nucleus accumbens (NA), hippocampal CA1, somatosensory cortex and dorsal cochlear nucleus (DCN). We compared mitochondria in the presynaptic terminals to those in the postsynaptic/dendritic compartments, and we focused on the shape and size of mitochondria. A common feature of mitochondria among the four brain regions is that presynaptic mitochondria generally are small and short, and most of them do not extend beyond presynaptic terminals. In contrast, the majority of postsynaptic/dendritic mitochondria are large and many of them spread through significant portions of the dendrites. Comparing among the brain areas, the cerebral cortex and DCN have even larger postsynaptic/dendritic mitochondria than the NA and CA1. Our analysis reveals that mitochondria in neurons are differentially sized and arranged according to their subcellular locations, suggesting a spatial organizing principle of mitochondria at the synapse.

In subsurface anoxic environments, microbial communities generally produce methane as an end-product to consume organic compounds. This metabolic function is a source of biogenic methane in coastal natural gas aquifers, submarine mud volcanoes, and methane hydrates. Within the methanogenic communities, hydrogenotrophic methanogens, and syntrophic bacteria are converting volatile fatty acids to methane syntrophically via interspecies hydrogen transfer. Recently, direct interspecies electron transfer (DIET) between fermentative/syntrophic bacteria and electrotrophic methanogens has been proposed as an effective interspecies metabolite transfer process to enhance methane production. In this study, in order to stimulate the DIET-associated methanogenic process at deep biosphere-aquifer systems in a natural gas field, we operated a bioelectrochemical system (BES) to apply voltage between an anode and a cathode. Two single-chamber BESs were filled with seawater-based formation water collected from an onshore natural gas well, repeatedly amended with acetate, and operated with 600 mV between electrodes for 21 months, resulting in a successful conversion of acetate to methane via electrical current consumption. One reactor yielded a stable current of ~200 mA/m2 with a coulombic efficiency (CE) of >90%; however, the other reactor, which had been incidentally disconnected for 3 days, showed less electromethanogenic activity with a CE of only ~10%. The 16S rRNA gene-based community analyses showed that two methanogenic archaeal families, Methanocalculaceae and Methanobacteriaceae, were abundant in cathode biofilms that were mainly covered by single-cell-layered biofilm, implicating them as key players in the electromethanogenesis. In contrast, family Methanosaetaceae was abundant at both electrodes and the electrolyte suspension only in the reactor with less electromethanogenesis, suggesting this family was not involved in electromethanogenesis and became abundant only after the no-electron-flow event. The anodes were covered by thick biofilms with filamentous networks, with the family Desulfuromonadaceae dominating in the early stage of the operation. The family Geobacteraceae (mainly genus Geoalkalibacter) became dominant during the longer-term operation, suggesting that these families were correlated with electrode-respiring reactions. These results indicate that the BES reactors with voltage application effectively activated a subsurface DIET-related methanogenic microbiome in the natural gas field, and specific electrogenic bacteria and electromethanogenic archaea were identified within the anode and/or cathode biofilms.

The visualization of cellular ultrastructure over a wide range of volumes is becoming possible by increasingly powerful techniques grouped under the rubric “volume electron microscopy” or volume EM (vEM). Focused ion beam scanning electron microscopy (FIB-SEM) occupies a “Goldilocks zone” in vEM: iterative and automated cycles of milling and imaging allow the interrogation of microns-thick specimens in 3-D at resolutions of tens of nanometers or less. This bestows on FIB-SEM the unique ability to aid the accurate and precise study of architectures of virus-cell interactions. Here we give the virologist or cell biologist a primer on FIB-SEM imaging in the context of vEM and discuss practical aspects of a room temperature FIB-SEM experiment. In an in vitro study of SARS-CoV-2 infection, we show that accurate quantitation of viral densities and surface curvatures enabled by FIB-SEM imaging reveals SARS-CoV-2 viruses preferentially located at areas of plasma membrane that have positive mean curvatures.

Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) tomography provides a stack of images that represent serial slices of the sample. These images are displaced relatively to each other, and an alignment procedure is required. Traditional methods for alignment of a 3D image are based on a comparison of two adjacent slices. However, such algorithms are easily confused by anisotropy in the sample structure or even experiment geometry in the case of porous media. This may lead to significant distortions in the pore space geometry, if there are no stable fiducial marks in the frame. In this paper, we propose a new method, which meaningfully extends existing alignment procedures. Our technique allows the correction of random misalignments between slices and, at the same time, preserves the overall geometrical structure of the specimen. We consider displacements produced by existing alignment algorithms as a signal and decompose it into low and high-frequency components. Final transformations exclude slow variations and contain only high frequency variations that represent random shifts that need to be corrected. The proposed algorithm can operate with not only translations but also with arbitrary affine transformations. We demonstrate the performance of our approach on a synthetic dataset and two real FIB-SEM images of natural rock.

One recent technical innovation in neuroscience is microcircuit analysis using three-dimensional reconstructions of neural elements with a large volume Electron microscopy (EM) data set. Large-scale data sets are acquired with newly-developed electron microscope systems such as automated tape-collecting ultramicrotomy (ATUM) with scanning EM (SEM), serial block-face EM (SBEM) and focused ion beam-SEM (FIB-SEM). Currently, projects are also underway to develop computer applications for the registration and segmentation of the serially-captured electron micrographs that are suitable for analyzing large volume EM data sets thoroughly and efficiently. The analysis of large volume data sets can bring innovative research results. These recently available techniques promote our understanding of the functional architecture of the brain.

Here we report on ultrastructural features of brain synapses in the fly Drosophila melanogaster and outline a perspective for the study of their functional significance. Images taken with the aid of focused ion beam-scanning electron microscopy (EM) at 20 nm intervals across olfactory glomerulus DA2 revealed that some synaptic boutons are penetrated by protrusions emanating from other neurons. Similar structures in the brain of mammals are known as synaptic spinules. A survey with transmission EM (TEM) disclosed that these structures are frequent throughout the antennal lobe. Detailed neuronal tracings revealed that spinules are formed by all three major types of neurons innervating glomerulus DA2 but the olfactory sensory neurons (OSNs) receive significantly more spinules than other olfactory neurons. Double-membrane vesicles (DMVs) that appear to represent material that has pinched-off from spinules are also most abundant in presynaptic boutons of OSNs. Inside the host neuron, a close association was observed between spinules, the endoplasmic reticulum (ER) and mitochondria. We propose that by releasing material into the host neuron, through a process triggered by synaptic activity and analogous to axonal pruning, synaptic spinules could function as a mechanism for synapse tagging, synaptic remodeling and neural plasticity. Future directions of experimental work to investigate this theory are proposed.

Plant roots are major transmitters of atmospheric carbon into soil. The rhizosphere, the soil volume around living roots influenced by root activities, represents hotspots for organic carbon (OC) inputs, microbial activity, and carbon turnover. Rhizosphere processes remain poorly understood and the observation of key mechanisms for carbon transfer and protection in intact rhizosphere microenvironments are challenging. We deciphered the fate of photosynthesis-derived OC in intact wheat rhizosphere, combining stable isotope labeling at field scale with high-resolution 3D-imaging. We used nano-scale secondary ion mass spectrometry and focus ion beam-scanning electron microscopy to generate insights into rhizosphere processes at nanometer scale. In immature wheat roots, the carbon circulated through the apoplastic pathway, via cell walls, from the stele to the cortex. The carbon was transferred to substantial microbial communuties, mainly represented by bacteria surrounding peripheral root cells. Iron oxides formed bridges between roots and bigger mineral particles, such as quartz, and surrounded bacteria in microaggregates close to the root surface. Some microaggregates were also intimately associated with the fungal hyphae surface. Based on these results, we propose a conceptual model depicting the fate of carbon at biogeochemical interfaces in the rhizosphere, at the forefront of growing roots. We observed complex interplays between vectors (roots, fungi, bacteria), transferring plant-derived OC into root-free soil and stabilizing agents (iron oxides, root and microorganism products), potentially protecting plant-derived OC within microaggregates in the rhizosphere.

Semithin sections are commonly used to examine large areas of tissue with an optical microscope, in order to locate and trim the regions that will later be studied with the electron microscope. Ideally, the observation of semithin sections would be from mesoscopic to nanoscopic scales directly, instead of using light microscopy and then electron microscopy (EM). Here we propose a method that makes it possible to obtain high-resolution scanning EM images of large areas of the brain in the millimeter to nanometer range. Since our method is compatible with light microscopy, it is also feasible to generate hybrid light and electron microscopic maps. Additionally, the same tissue blocks that have been used to obtain semithin sections can later be used, if necessary, for transmission EM, or for focused ion beam milling and scanning electron microscopy (FIB-SEM).

The transmission cycle of chikungunya virus (CHIKV) requires that mosquito vectors get persistently infected with the virus, following its oral acqsuisition from a vertebrate host. The mosquito midgut is the initial organ that gets infected with orally acquired CHIKV. Following its replication in the midgut epithelium, the virus exits the midgut and infects secondary tissues including the salivary glands before being transmitted to another host. Here, we investigate the pattern of CHIKV dissemination from the midgut of Aedes aegypti at the ultrastructural level. Bloodmeal ingestion caused overstretching of the midgut basal lamina (BL), which was disrupted in areas adjacent to muscles surrounding the midgut as shown by scanning electron microscopy (SEM). Using both transmission electron microscopy (TEM) and focused ion beam scanning electron microscopy (FIB-SEM) to analyze midgut preparations, mature chikungunya (CHIK) virions were found accumulating at the BL and within strands of the BL at 24–32 h post-infectious bloodmeal (pibm). From 48 h pibm onwards, virions no longer congregated at the BL and became dispersed throughout the basal labyrinth of the epithelial cells. Ingestion of a subsequent, non-infectious bloodmeal caused mature virions to congregate again at the midgut BL. Our study suggests that CHIKV needs a single replication cycle in the midgut epithelium before mature virions directly traverse the midgut BL during a relatively narrow time window, within 48 h pibm.

Premise of the study: Focused ion beam–scanning electron microscopy (FIB-SEM) combines the ability to sequentially mill the sample surface and obtain SEM images that can be used to create 3D renderings with micron-level resolution. We have applied FIB-SEM to study Arabidopsis cell architecture. The goal was to determine the efficacy of this technique in plant tissue and cellular studies and to demonstrate its usefulness in studying cell and organelle architecture and distribution. Methods: Seed aleurone, leaf mesophyll, stem cortex, root cortex, and petal lamina from Arabidopsis were fixed and embedded for electron microscopy using protocols developed for animal tissues and modified for use with plant cells. Each sample was sectioned using the FIB and imaged with SEM. These serial images were assembled to produce 3D renderings of each cell type. Results: Organelles such as nuclei and chloroplasts were easily identifiable, and other structures such as endoplasmic reticula, lipid bodies, and starch grains were distinguishable in each tissue. Discussion: The application of FIB-SEM produced 3D renderings of five plant cell types and offered unique views of their shapes and internal content. These results demonstrate the usefulness of FIB-SEM for organelle distribution and cell architecture studies.